In organisms ranging from single-celled yeasts to mammals, genetically identical cells exhibit variable cell division cycle times even when growing in the same environment. The sources and benefits of variability in a process such as the cell cycle that is wired for accuracy are unknown. In particular, it is not understood whether cell cycle timing variability is purely stochastic or whether it may be regulated as part of the design of cell cycle circuits. Variability in other cell processes has been shown to be beneficial, so cell cycle variability may in fact be an adaptive trait. The multinucleate, filamentous fungus, Ashbya gossypii, is a unique model system to study cell cycle timing variability because nuclei divide asynchronously within a common cytoplasm. Such asynchrony in a syncytium requires variable timing and nuclear autonomy in cell cycle signaling. As all proteins are translated in a common cytoplasm, it is mysterious how multiple, out of sync, cell cycle oscillators can coexist. We are taking advantage of the asynchronous division cycle in this model system to discover whether variability is programmed into the cell cycle and to learn how nuclear autonomy can be established. Knowledge of the molecular basis for variability is necessary for a complete understanding of cell cycle control and the pathologies influenced by a misregulated cell cycle. Population level variability in cell cycle decisions can impact processes as diverse as fungal pathogenesis and tumor cell behavior, and may be a factor influencing the efficacy of pharmacological treatments. While some cell-to-cell variability can be attributed to molecular noise in transcription, it is certain that other, as yet unidentified, cellular reservoirs of non-genetic individuality exist. In this proposal, we combine live cell imaging with computational and molecular genetic approaches to identify sources of variability in the cell cycle and determine how nuclear autonomy is established. With this model fungal system, we are well positioned to identify conserved sources of cell cycle variability and learn how cell signaling processes can be insulated within a common cytoplasm. The specific aims of the project are: 1) To determine whether variability in G1 duration is stochastic or regulated. 2) To test the hypothesis that nuclear size controls cell cycle timing and variability. 3) To test the hypothesis that spatially restricted protein movement creates nuclear autonomy. Timing variability exists in nearly all cell division cycles and knowing the basis of heterogeneity is essential for a complete understanding of the cell cycle. In this project, we will determine if timing variability is programmed in the cell division cycle, learn how nuclear size controls timing and how the cytoplasm can be functionally compartmentalized to maintain asynchrony.